The eukaryotic ssDNA binding protein Replication Protein A (RPA) is required for nearly all DNA transactions in the cell including replication, damage response, and repair. RPA is a heterotrimer of 70, 32, and 14 kDa subunits composed of modular domains of varied function. The 70N and 32C protein interaction domains of RPA are involved in the recruitment of DNA damage response and DNA repair proteins to replication forks stalled at DNA damage sites. Although the RPA scaffold is essential to DNA damage response and DNA repair, the molecular mechanism of how the 70N and 32C domains maneuver components of the protein machinery remains elusive. Probe molecules capable of selectively inhibiting RPA70N or RPA32C interactions hold great promise as powerful reagents to more thoroughly dissect RPA70N and RPA32C function in the processing of DNA. Here, we propose using fragment based design via Structure Activity Relationship (SAR) by NMR to develop small molecule inhibitors of the protein interaction interfaces of RPA70N and RPA32C. Aim 1 focuses on RPA70N inhibitors and builds on promising preliminary data that have identified an initial pharmacophore from a library of molecular fragments, and the site to which these molecules bind. We will use binding affinities for RPA70N as well as structures of ligand complexes to guide the design of optimized probes based on these first site binders and begin a second screen to identify ligands that bind in a different site. Aim 2 will follow on this SAR by NMR fragment-based discovery approach to develop inhibitors of the RPA32C protein binding interface. Aim 3 will employ biophysical methods to characterize the extent of inhibition by the probes developed in Aims 1 and 2, using peptides derived from the RPA-binding domains of the proteins ATR Interaction Protein (ATRIP) and SWI/SNF-related Matrix-associated Actin-dependent Regulator of Chromatin subfamily A-Like protein 1 (Smarcal1). Once validated, these compounds will be used in functional assays by our collaborators to elucidate the mechanisms of action in the corresponding DNA damage response and DNA repair pathways. These compounds may also serve as potential leads for translation of this basic research into the development of therapeutic agents. PUBLIC HEALTH RELEVANCE: This study will develop probe molecules capable of inhibiting the function of select RPA domains in order to further understand of how RPA choreographs the assembly and disassembly of DNA damage response and repair complexes at sites of DNA damage. Once obtained, these probes will serve as powerful reagents for functional assays by our collaborators to elucidate the mechanisms of action in the DNA damage response and DNA repair pathways. Additionally, our study will provide a clearer structural understanding of the translational potential of these inhibitors towards the development of new cancer therapeutics.